Membrane-docking loops of the cPLA2 C2 domain: detailed structural analysis of the protein-membrane interface via site-directed spin-labeling

Abstract

C2 domains are protein modules found in numerous eukaryotic signaling proteins, where their function is to target the protein to cell membranes in response to a Ca(2+) signal. Currently, the structure of the interface formed between the protein and the phospholipid bilayer is inaccessible to high-resolution structure determination, but EPR site-directed spin-labeling can provide a detailed medium-resolution view of this interface. To apply this approach to the C2 domain of cytosolic phospholipase A(2) (cPLA(2)), single cysteines were introduced at all 27 positions in the three Ca(2+)-binding loops and labeled with a methanethiosulfonate spin-label. Altogether, 24 of the 27 spin-labeled domains retained Ca(2+)-activated phospholipid binding. EPR spectra of these 24 labeled domains obtained in the presence and absence of Ca(2+) indicate that Ca(2+) binding triggers subtle changes in the dynamics of two localized regions within the Ca(2+)-binding loops: one face of the loop 1 helix and the junction between loops 1 and 2. However, no significant changes in loop structure were detected upon Ca(2+) binding, nor upon Ca(2+)-triggered docking to membranes. EPR depth parameters measured in the membrane-docked state allow determination of the penetration depth of each residue with respect to the membrane surface. Analysis of these depth parameters, using an improved, generalizable geometric approach, provides the most accurate picture of penetration depth and angular orientation currently available for a membrane-docked peripheral protein. Finally, the observation that Ca(2+) binding does not trigger large rearrangements of the membrane-docking loops favors the electrostatic switch model for Ca(2+) activation and disfavors, or places strong constraints on, the conformational switch model.

Figure 1

Structure of the C2 Domain of cPLA₂. (A) The crystal structure of the C2 domain of cPLA₂ is shown (22), with eight β -strands depicted by ribbons and two Ca²⁺ ions shown as spheres. (B) Enlarged view of the three Ca²⁺-binding loops (CBLs). Figure was generated in Molscript (56).

Figure 2

Effects of Ca²⁺ binding and membrane docking on EPR spectra. (A) Shown are EPR X-band spectra for spin-labeled C2 domains acquired in the presence of 5 mM EDTA (thin line) or 2 mM CaCl₂ (heavy line) for 12 representative modified proteins. (B) Shown are EPR spectra for spin-labeled C2 domains acquired in the presence of 2 mM CaCl₂ without (thin line) or with (heavy line) 40 mM PC/PS membranes for 12 representative modified proteins. Samples contained 20–200 μM protein in 20 mM HEPES, 100 mM KCl, pH 7.4, and spectra were acquired at 23 °C.

Figure 3

Scaled mobilities of C2 domain spin-labels. The scaled mobility of a specific spin-label position was determined from the peak-to-peak line width of its central transition (δ). This line width was compared to standard line widths for highly mobile and immobile spin-labels to calculate the scaled mobility (M_s) using the relationship M_s = δ⁻¹ − δ_i⁻¹)/(δ_m ⁻¹ − δ_i⁻¹), where δ_i= 8.4 G and δ_m = 2.1 G are the standard calibration line widths (39). (A) Scaled mobility parameters for EPR spectra of labeled C2 domains without Ca²⁺ (open), with Ca²⁺ (hatched), and with both Ca²⁺ and phospholipid membranes (closed). (B) Fractional change in scaled mobility, calculated as [(M_s(Ca²⁺) − M_s(apo))/M_s(apo)] (open) or as [(M_s(PL) − M_s(Ca²⁺))/M_s(Ca²⁺)] (closed). EPR parameters and sample conditions are as in Figure 2.

Figure 4

Continuous-wave power saturation depth parameters for the three Ca²⁺-binding loops. (A) Ca²⁺-binding loop I, (B) Ca²⁺-binding loop II, and (C) Ca²⁺-binding loop III. The depth parameter Φ is calculated as the log ratio of spin-label collision rates with dioxygen and with NiEDDA (Φ = ln[Π(O₂)/ Π(NiEDDA)]). EPR spectra were single 2 min scans, with sample conditions as in Figure 2. Measured values reflect the error-weighted average of duplicate experiments, using errors propagated from measurements of P_1/2 and H_pp (see Materials and Methods). Errors in the average values shown were calculated as the inverse square root of the sum of the inverse squares of errors in individual measurements [error of mean = (Σ(individual error)⁻²)^−1/2] (42).

Figure 5

Depth parameter vs distance plots for spin-labeled lipids and C2 domains. Depth parameters for spin-labeled lipids (open squares) and spin-labeled proteins with positive depth parameters (closed circles) are plotted as a function of distance from membrane phosphates. (A) Depth parameter–distance plot for the model calculated with all spin-labels in a g+g+ conformation, focusing on spin-labels with positive depth parameters. Shown is the best-fit straight line for these data points. (B) Depth parameter–distance plot for the model calculated with the conformations of labels at V97R1 and M98R1 adjusted to g–g+ and tg+ conformations, respectively, while maintaining all other labels in a g+g+ conformation. Shown is the best-fit straight line for these data points, again focusing on spin-labels with positive depth parameters. (C) Plot including points for protein labels with negative depth parameters (open circles). Shown is the best-fit straight line from panel B that accurately describes spin-labels possessing positive depth parameters, located in a region ranging from the hydrocarbon phase to the deeper half of the headgroup layer (41, 57). Also shown is the best-fit hyperbolic tangent function that provides a better model for the negative depth parameters observed in a region ranging from the aqueous half of the headgroup layer to the bulk aqueous phase (32).

Figure 6

Orientation and depth of the C2 domain of cPLA₂ with respect to a membrane surface. The crystal structure of the C2 domain of cPLA₂ (22) is represented in cyan ribbons, with two Ca²⁺ ions shown as yellow spheres. The phosphate plane of the membrane is depicted as a solid line, such that the glycerol backbone and hydrocarbon core regions of the membrane lie above the line. Protein spin-labels oriented in their final optimized conformations are colored according to their measured depth parameters, with positive depth parameters indicated by increasing red and negative depth parameters indicated by increasing blue. The figure was generated using Insight2000 (Accelrys).

Figure 7

Modeled depth and orientation of the cPLA₂ C2 domain on a simulated membrane bilayer. The crystal structure of the C2 domain of cPLA₂ (22) with the final modeled spin-label conformations is superimposed on a simulated membrane bilayer (59, 60). The β-strands of the protein are shown in cyan, with two Ca²⁺ ions in yellow spheres and protein spin-labels in green. The membrane is depicted with hydrocarbon chains in black and headgroup atoms and water molecules shown in gray. This schematic representation was generated in Molscript (56) using a modified protein crystal structure generated in Insight2000 (Accelrys).

Figure 8

Rotations and translation used to compare different docking models. A C2 domain is depicted as a solid rectangle, with the three α-carbons used to define the molecular axes shown as circles and the molecular x′- and z′-axes shown as rods (see text). The membrane bilayer is shown in yellow. The universal starting position places the z′-axis normal to the membrane surface, such that the calibration β-sheet is perpendicular to the membrane, and fixes a calibration atom at a standard distance from the membrane. To transform this starting position into the final modeled position, the domain is rotated about the x′-axis, then rotated about the z′-axis to produce the final angular orientation, and subsequently is translated along the membrane normal to generate the final membrane penetration depth. Rotations and translations needed to reproduce the published docking models for the cPLA₂, PKCα, and SytIa C2 domains are compared in Table 3.